A polylinker approach to reductive loop swaps in modular polyketide synthases.
Multiple versions of the DEBS 1-TE gene, which encodes a truncated bimodular polyketide synthase (PKS) derived from the erythromycin-producing PKS, were created by replacing the DNA encoding the ketoreductase (KR) domain in the second extension module by either of two synthetic oligonucleotide linkers. This made available a total of nine unique restriction sites for engineering. The DNA for donor "reductive loops," which are sets of contiguous domains comprising either KR or KR and dehydratase (DH), or KR, DH and enoylreductase (ER) domains, was cloned from selected modules of five natural PKS multienzymes and spliced into module 2 of DEBS 1-TE using alternative polylinker sites. The resulting hybrid PKSs were tested for triketide production in vivo. Most of the hybrid multienzymes were active, vindicating the treatment of the reductive loop as a single structural unit, but yields were dependent on the restriction sites used. Further, different donor reductive loops worked optimally with different splice sites. For those reductive loops comprising DH, ER and KR domains, premature TE-catalysed release of partially reduced intermediates was sometimes seen, which provided further insight into the overall stereochemistry of reduction in those modules. Analysis of loops containing KR only, which should generate stereocentres at both C-2 and C-3, revealed that the 3-hydroxy configuration (but not the 2-methyl configuration) could be altered by appropriate choice of a donor loop. The successful swapping of reductive loops provides an interesting parallel to a recently suggested pathway for the natural evolution of modular PKSs by recombination.
Kellenberger
Laurenz
L
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB21GA, United Kingdom.
Galloway
Ian S
IS
Sauter
Guido
G
Böhm
Günter
G
Hanefeld
Ulf
U
Cortés
Jesús
J
Staunton
James
J
Leadlay
Peter F
PF
D000595:Amino Acid Sequence; D055162:Biocatalysis; D004247:DNA / Q000235:genetics*; D017665:Hydroxyl Radical; D007783:Lactones / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D010084:Oxidation-Reduction; D010455:Peptides / Q000235:genetics; D048630:Polyketide Synthases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D016930:Saccharopolyspora / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity
D007783:Lactones; D009841:Oligonucleotides; D010455:Peptides; D017665:Hydroxyl Radical; D048630:Polyketide Synthases; D004247:DNA